In the brain, key focal points for control of metabolic function and feeding behavior are melanocortin receptors MC3R and MC4R. These receptors respond to two ligands, alpha-melanocyte stimulating hormone and Agouti-related protein (AgRP), which act in opposite ways to promote negative and positive energy balance, respectively. The goal of this program is to understand the molecular basis of MCR regulation. Work from the last funding period uncovered new mechanistic features of AgRP processing, solved the NMR structure of the homologous agouti signaling protein (ASIP) found in the skin, and identified 2-defensins, released by the immune system, as a new class of MCR ligands. The next phase of this research will build on these advances with the following four projects. 1) The AgRP N-terminal domain greatly inhibits its affinity for MCRs. NMR, pharmacology and collaborative in vivo studies, will be used to test the hypothesis that a partially conserved acidic domain docks to a C-terminal loop thus occluding receptor docking. 2) 2-Defensins are not homologous to AgRP or ASIP, but comparison of their respective structural features identifies specific loops that may target MCRs. Structure- function studies will uncover the structural basis for 2-defensin antagonism of MCRs. 3) Although published work identifies 2-defensin 3 as a ligand to MC1R and MC4R, preliminary results show that other defensins bind as well. A screen across the family of 2-defensins will be performed to identify additional MCR interactions, thus pointing to potential new 2-defensin functions. 4) 2-Defensins are challenging to fold in vitro, thus confounding important functional investigations. Human ASIP presented similar difficulties that were overcome by the PI's lab using protein engineering. Analogous strategies will be used with members of the 2-defensin family to advance the understanding of the defensin fold and to produce useful protein for pharmacological and structural studies.